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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 10  |  Page : 634-643

Knockdown of alpha-fetoprotein expression inhibits HepG2 cell growth and induces apoptosis


1 Department of General Surgery, Gansu Provincial Hospital, Lanzhou 730000, Gansu, China
2 Department of Infectious Disease, The First Hospital of Lanzhou University, Lanzhou 730000, Gansu, China
3 Department of General Surgery, The People's Hospital of Baoji City, Baoji 721000, Shaanxi, China

Date of Web Publication24-Sep-2018

Correspondence Address:
Peng Gao
Department of General Surgery, Gansu Provincial Hospital, Lanzhou 730000, Gansu
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.180681

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 > Abstract 


Aims: To explore the biological roles of alpha-fetoprotein (AFP), a tumor-associated antigen in human hepatocellular carcinoma (HCC).
Materials and Methods: After knockdown of AFP in HepG2 cells by transfection of specific Stealth™ RNAi, the expression of AFP were detected by reverse transcription polymerase chain reaction at mRNA level and by enzyme-linked immunosorbent assay at the protein level. Then, the effect of silenced AFP on cell proliferation was assessed by dimethylthiazolyl-2,5-diphenyl-tetrazolium bromide assay, and apoptosis assessment with Hoechst33258 and flow cytometry (double stain with fluorescein isothiocyanate/propidium iodide), the roles of AFP in the cell cycle regulation were assessed by flow cytometry. We also detected the expression of some key proteins related to apoptosis pathway by Western immunoblot analysis.
Results: After the transfection for 48 h, the expression of AFP gene was almost abolished, the cell proliferation was inhibited by 47.61%, the number of cells undergoing early apoptosis was significantly increased to 59.47%; cell cycle was arrested with the increase of G0/G1 phase cells from 45.3% to 58.4%. Inhibition of AFP expression also results in decreasing of transforming growth factor-β (TGF-β), mutant P53 expression, and increasing of Bax/Bcl-2 ratio, activation of caspase-3.
Conclusions: The results suggest that AFP may positively regulate cell proliferation by enhancing the apoptosis resistance via effect on TGF-β and p53/Bax/caspase-3 signaling pathway in HepG2 cells. As such, the knockdown of AFP gene should be further investigated in vivo as a novel approach to HCC treatment.

Keywords: Alpha-fetoprotein, apoptosis, hepatocellular carcinoma, RNA interference, transforming growth factor-β


How to cite this article:
Yang X, Chen L, Liang Y, Si R, Jiang Z, Ma B, Gao P. Knockdown of alpha-fetoprotein expression inhibits HepG2 cell growth and induces apoptosis. J Can Res Ther 2018;14, Suppl S3:634-43

How to cite this URL:
Yang X, Chen L, Liang Y, Si R, Jiang Z, Ma B, Gao P. Knockdown of alpha-fetoprotein expression inhibits HepG2 cell growth and induces apoptosis. J Can Res Ther [serial online] 2018 [cited 2019 Sep 20];14:634-43. Available from: http://www.cancerjournal.net/text.asp?2018/14/10/634/180681




 > Introduction Top


Hepatocellular carcinoma (HCC) is one of the most common solid tumors, rated fifth in incidence and the third in mortality worldwide.[1] Traditionally, the care of patients with HCC has been undertaken by hepatobiliary surgeons (surgical resection or transplantation), interventional radiologists (percutaneous ablation or transarterial embolization), and oncologists (systemic chemotherapy). Despite therapeutic advances, there has not been a significant improvement in the overall survival of patients who have hepatocellular cancers in the last two decades. Most published studies of systemic chemotherapy report lower response rates of 0–25%, and systemic chemotherapy has never been shown to prolong survival in patients with HCC.[2] Although some major etiological agents have been identified, the molecular pathogenesis of HCC remains unclear,[3] and new approaches are critically needed for the significant reduction in HCC mortality due to the failure of conventional chemotherapy. Therefore, further study of molecular mechanism of HCC pathogenesis and exploration of new targets for molecular HCC treatment are very important at present.

Alpha-fetoprotein (AFP) is the major serum protein synthesized during fetal life and its content decreases rapidly during early postnatal life.[4] Since its relationship to cancer was reported in the mid-1960s, this tumor-associated fetal protein has demonstrated clinical utility both as a tumor marker and a birth defect screening agent.[5] Although the physicochemical and structural properties of this 70-kDa glycoprotein have been extensively described, only in vitro functional roles of this oncofetal protein have been ascertained to date. Such physiological properties of the oncofetal protein have encompassed mainly ligand carrier/transport functions and modulation of in vitro immune response assays.[6],[7] In the last decade, the growth regulatory properties of AFP have aroused interest as a result of studies involving ontogenetic and oncogenic growth in both cell cultures and animal models. A myriad of studies have now documented that AFP is capable of regulating growth in ovarian, placental, uterine, hepatic, phagocytic, bone marrow, and lymphatic cells,[8],[9] in addition to various neoplastic cells.[10],[11] Up to now, a lot of research results provide further support to the idea that AFP is a bifunctional protein acting in cellular proliferation and apoptosis of HCC.[10],[12],[13],[14],[15],[16],[17],[18],[19],[20] In the authors' view, AFP should no longer be considered merely a fetal form of albumin only to be employed as a marker for cancer and fetal disorders; rather, AFP should now be considered as a possible direct or indirect factor associated with the regulation of growth, apoptosis in oncogenic growth processes. However, the biological role of this major embryonic serum protein is still unknown although numerous speculations have been made.[21] Our previous works have demonstrated that silencing expression of AFP can inhibits cell growth and induces apoptosis through the p53/Bax/cytochrome c/caspase-3 apoptosis signaling pathway in Huh7 cells.[22] The aim of the study was to further explore this problem in HepG2 cells.

Small interfering RNA (siRNA) have been shown to inhibit the expression of a corresponding target gene in mammals,[23] where these siRNA molecules are separated into single strands and incorporated into the RNA-induced silencing complex, which then cleaves the corresponding cellular mRNA, indicating that RNAi may be served as a powerful technology to specifically block the expressing of target genes.[24],[25],[26] In this study, we down-regulated AFP expression by Stealth RNAi in an HCC cell line HepG2 with relatively high AFP expression and evaluated the effect of decreased AFP expression on cell apoptosis and biological behaviors in HepG2 cell. We also explored the possible molecular mechanisms that underlie the inhibition of proliferation and apoptosis induction by knockdown of AFP in HepG2 cells. The results of this study suggest that AFP may positively regulate cell proliferation by enhancing the apoptosis resistance via affect on TGF-b and p53/Bax/caspase-3 signaling pathway in HepG2 cells. As such, the knockdown of AFP gene should be further investigated in vivo as a novel approach to HCC treatment.


 > Materials and Methods Top


Design and synthesize of Stealth RNAi targeting alpha-fetoprotein gene sequence

According to the siRNA design guidelines,[27],[28] three different RNAi target sequences were selected corresponding to nucleotides152–176 (HSS303-Stealth RNAi), 245–269 (HSS304-Stealth RNAi), and 546–570 (HSS305-Stealth RNAi) of the human AFP mRNA (GenBank Accession No. NM001134). The three 5-nucleotide modified synthetic stealth RNAi targeting AFP were customarily synthesized by Invitrogen Inc., without overhanging at the 3'end, and Stealth RNAi duplexes whose GC content is similar to that of each duplex siRNA (low GC content) from Invitrogen Inc., were used as negative control. Sequences of the three synthesized oligonucleotides are HSS303: Sense5'-UAAACUUAUCUCUGCAGUACAUUGG-3', anti-sense5'-CCAAUGU ACUGGAGAGAUAAGUUUA-3'; HSS304: Sense5'-AAUUGCAGUCAAUGCAUCUUUCACC-3', anti-sense 5'-GGUGAAAGAUGCAUUGACUGCAAUU-3'; and HSS305: Sense5'-CAUACAGGAAGGGAUGCCUUCUUG C-3', anti-sense 5'-GCAAGAAGGCAUCCCUUCCUGUAUG-3'. These target sequences were submitted to a BLAST search to ensure that only the AFP gene was targeted.

Cell culture and transfection

HepG2 cells were maintained and grown as a monolayer in Roswell Park Memorial Institute (RPMI) Medium 1640 (Invitrogen Inc., USA) supplemented with 10% heat-inactivated newborn calf serum, penicillin G (100 units/ml), and streptomycin (100 units/ml) in humidified incubator containing 50 mL/L CO2 at 37°C. The cells at about 70% confluence were used for all the experiments in this study.

Synthesized Stealth RNAi against AFP was transfected into HepG2 cells using the Lipofectamine™ RNAi Max transfection agent (Invitrogen Inc., USA) according to the manufacturer's protocol. Meanwhile, the negative control was used to prevent induction of nonspecific cellular events caused by the introduction of the oligonucleotide into cells. The transfection efficiency of each duplex siRNA was confirmed by using BLOCK-iT™ Alexa Fluor® Red Fluorescent (Invitrogen Inc., USA) according to the manufacturer's instructions. The duplex siRNA is not homologous to any other known genes. Uptake of the Fluorescent Oligo, which correlates strongly with uptake of Stealth™ RNAi, was used to measure transfection efficiency for optimization of RNAi transfection. Reverse transfection was applied to deliver Stealth™ RNAi, fluorescent oligo or negative control duplexes into HepG2 cells. Briefly, the complexes were prepared inside the wells, then cells and medium were added and incubated at 37°C in a CO2 incubator until assayed for gene knockdown. Cell number added per well was optimized such that 24 h after plating, cell confluence is 30–50%.

Reverse transcription polymerase chain reaction-assess Stealth RNAi effects

HepG2 cells (3 × 105) were seeded onto six-well plates. Total RNA was extracted using Trizol reagent (Invitrogen Inc., USA) following the manufacturer's instruction at 48 or 72 h after Stealth RNAi transfection. First strand cDNA synthesis and amplification were performed using the two-step real-time reverse transcription-polymerase chain reaction (RT-PCR) kit (TakaRa, Japan). Quantitative PCR amplifications were performed with a 7000 Sequence Detection System (Applied Biosystems, USA). Reactions were carried out in a 25 μl reaction volume containing 12.5 μl of 2 × SYBR® Premix Ex Taq™ (TakaRa, Japan). β-actin was used as an internal control. The sequences of primers are AFP sense 5'-GGAAGTCTGCTTTGCTG AAGA-3'and anti-sense 5'-CACACCGAATGAAAGACTCGT-3'(GenBank Accession No. NM001134.1), β-actin sense5'-GCAAGCAGGAGTATGACGAGT-3'and anti-sense 5'-CTGCGCAAGTTAGGTTTTGTC-3' (GenBank, Accession No.NM031144) (Shanghai Sangon, China). Thermal cycle conditions: 95°C for 10 s, followed by forty cycles of 95°C 5 s, 60°C 30 s. The ΔCt of each group was calculated by formula: ΔCt = Ct AFP-Ct β-actin. ΔΔCt was calculated by ΔCt treated-ΔCt control. The fold-change for AFP expression levels of the treated groups were calculated using 2-ΔΔCt.

Alpha-fetoprotein protein detection by enzyme-linked immunosorbent assay

The supernatant of HepG2 cells was collected after transfection with Stealth RNAi or negative control for 48 or 72 h. AFP proteins in the supernatant of HepG2 cell was measured using a commercially available human AFP enzyme-linked immunosorbent assay (ELISA) kit (Autobio Co., LTD, China) according to the manufacturer's instructions. The optical absorbance of the samples was measured at 450 nm using a microplate reader from Bio-Rad, USA. Standards of defined concentrations were run in each assay allowing the construction of a calibration curve by plotting absorbance versus concentration. The AFP protein concentrations in the supernatant were then calculated from this calibration curve and expressed as ng/ml.

Dimethylthiazolyl-2,5-diphenyl-tetrazolium bromide assay

After reverse transfection of HepG2 cells with Stealth RNAi or negative control duplexes in 96-well plates, dimethylthiazolyl-2,5-diphenyl-tetrazolium bromide (MTT) was added (24 μl/well of 5 g/L solution in phosphate buffered saline [PBS]) at either 24 h, 48 h, or 72 h. The plate was then incubated at 37°C for 4 h; the reaction was stopped by addition of 180 μl dimethyl sulfoxide. The crystallized MTT was dissolved and the absorbance was measured using an ELISA reader (Victor Co., Finland) at 490 nm wavelength. All samples were assayed repeatedly in six wells. The percentage of cell proliferation for each group with different treatments was calculated using the formula: Cell proliferation ratio = (A490 treatment/A490 control) ×100%.

Apoptosis assessment with Hoechst33258 and flow cytometry (double stain with fluorescein isothiocyanate/propidium iodide)

The cells undergoing apoptosis were visualized with Hoechst 33258 staining.[29] Briefly, 48 h or 72 h after stealth RNAi transfection, HepG2 cells plated on glass microscope slides in six-well plates were fixed with 70% ethanol for 10 min followed by staining with Hoechst 33258 (Beyotime, China, 500 μl/well) at room temperature in dark for 5 min. The cells were then washed twice with PBS, examined and immediately photographed under a fluorescence microscope (Nikon Corporation, Chiyoda-ku, Tokyo, Japan) with an excitation wavelength of 350 nm. Apoptotic cells were defined on the basis of nuclear morphology changes, such as chromatin condensation and fragmentation.

To quantitatively analyze the effects of Stealth RNAi on cell apoptosis, HepG2 cells were transfected with Stealth RNAi against AFP or negative control RNAi in culture flasks. After 48 or 72 h, cells were harvested by trypsinization and rinsed with cold PBS twice. After centrifugation (4°C, 1000 ×g) for 10 min, cells were resuspended in 200 μl binding buffer and then treated with 10 μl Annexin V-fluorescein isothiocyanate (FITC) and 5 μl propidium iodide (PI) (Sigma, USA) for 15 min at room temperature. Flow cytometric analysis of cells was performed with an EpicsXL Coulter flow cytometer (BECKMAN-COULTER, USA). FITC−/PI− cells were counted as normal control. The quantity of FITC+/PI− cells corresponded to early apoptosis, while that of FITC+/PI+ cells corresponded to late apoptosis or secondary necrosis.[30]

Cell cycle assessment with flow cytometry

To analyze the effect of knockdown of AFP on cell cycle progression, HepG2 cells were transfected with Stealth RNAi or negative control into culture flasks, respectively, and cultured in RPMI-1640 supplemented with 10% heat-inactivated newborn calf serum without antibiotic, then each group cells (1 × 106) were collected and washed with ice-cold PBS, and fixed in 70% ethanol overnight at 4°C for 48 h. The fixed cells were pelleted, washed with PBS, and resuspended in PBS containing 0.1 mg/mL of PI (Sigma, USA). DNA content profile of a given population was determined by flow cytometer (Becton Dickinson, USA).

Protein extraction and Western immunoblot analysis

HepG2 cells (5 × 105) were seeded into culture flasks. After 72 h transfection, cells were washed twice with cold PBS, and each culture flask was treated with 300 μl RIPA buffer (50 mM Tris-HCl, pH 8.0,150 mM NaCl; 1% NP-40,0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 2 mM ethylenediaminetetraacetic acid, supplemented with protease inhibitor 1 mM phenylmethanesulfonyl fluoride, all from Sigma) and incubated for 30 min on ice. Unbroken cells and large debris were removed by centrifugation at 12000 ×g for 5 min at 4°C, the resulting supernatants were saved at −70°C until further analysis.

Protein concentration was determined using the bicinchoninic acid protein assay reagent (Applygen Technologies Inc., China). Equal amounts of protein (20 μg) were separated on a 12–15% SDS-polyacrylamide gel (Bio-Rad, USA) and transferred to a PVDF membrane (Milipore, USA). The SDS-PAGE molecular weight markers (Bio-Rad, USA) verified the correct location of the visualized bands. Membranes were blocked in 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature and incubated with the primary antibody (dilution 1:400–2000) in TBST containing 1% milk overnight at 4°C. The primary antibodies were anti-AFP (Biosynthesis Biotechnology Co., LTD, China), transforming growth factor-β (TGF-β), Bcl-2, Bax, caspase-3 (Santa Cruz Biotech, USA), respectively. After four times washing with TBST, the blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1:2500, Santa Cruz Biotech, USA) at room temperature for 2 h. Immunocomplexes were visualized by incubation of the filters with enhanced chemiluminescence reagent (Chemicon International, USA) and exposure on X-ray film. To confirm equal protein loading, membranes were reprobed with a 1:2000 dilution of an anti-β-actin antibody (Santa Cruz Biotech Inc., USA). Densitometric analyses were performed using Scion Image software (National Institutes of Health, USA).

Statistical analysis

Data were expressed as a mean ± standard deviation (SD) one-way ANOVA followed by Bonferroni correction was used to compare the data among three or more groups, a Student's t-test was also used. All statistical analyses were performed using the SPSS 15.0 software package for Windows (SPSS Inc., Chicago, IL, USA) and a value of P < 0.05 was considered significant.


 > Results Top


Transfection efficiency and conditions

In the present study, stealth RNAi was used to determine the effect of knockdown of AFP gene on cell growth and apoptosis. To optimize conditions for siRNA knockdown, we first determined transfection efficiency using BLOCK-iT™ Alexa Fluor® Red Fluorescent control. Twenty-four hours after reverse transfection, HepG2 cells were observed using a fluorescent microscope. The transfection efficiency was evaluated by counting the red fluorescent cells, which was over 95% when using 20nM/L final concentration of oligo duplex and 1.5 × 105/ml HepG2 cells [Figure 1]. Therefore, we used 20nM/L siRNA and 1.5 × 105/ml HepG2 cells to perform subsequent experiments. In the cocktail group, the final concentration for each of the three stealth RNAi was 10nM/L.
Figure 1: Determine the transfection efficiency with BK-iTTM ALexa Fluor@ Red Fluorescent Control. Cells take up the Alexa Fluor Red Fluorescen control oligo 24 h after stealth RNAi transfection (a, ×100; b, ×400). A normal morphology was seen in the bright-field image (c, ×100; d, ×400)

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Stealth RNAi specifically reduces alpha-fetoprotein expression both at mRNA and protein levels in HepG2 cells

Following transfection of HepG2 cells with the stealth siRNA targeting AFP, we measured steady-state mRNA levels by real-time RT-PCR. Results showed an obvious decrease of AFP mRNA levels with the AFP RNAi while the levels of the housekeeping gene β-actin remained relatively unchanged. Of the three stealth RNAi against AFP, HSS304 showed a more potent suppression of AFP mRNA expression than HSS303 or HSS305 did. Comparatively, the cocktail of the three stealth RNAi showed the strongest suppression while transfection of negative control had no effect on AFP mRNA expression. Quantification analysis revealed that HSS304 reduced AFP mRNA by 81.9% and 87.6% of the blank control at 48 h and 72 h after stealth RNAi transfection, respectively. The reduction rates of the RNAi cocktail were 90.1% (48 h) and 92.5% (72 h) of the blank control, whereas HSS303, HSS305, and negative control transfected group were 55.6%, 69.5%, and 12% at 48 h and 67.2%, 77.3%, and 14% at 72 h after stealth RNAi transfection, respectively. There was a significant difference between blank control and the stealth RNAi groups ([Figure 2]a, P < 0.01).
Figure 2: Expression of alpha-fetoprotein mRNA and protein at 48 h, 72 h after transfection. (a) Relative expression of alpha-fetoprotein mRNA level (% blank control) analyzed by reverse transcription polymerase chain reaction *P < 0.01, vs. blank group; (b) expression of alpha-fetoprotein proteins in supernatant of HepG2 cells were detected by enzyme-linked immunosorbent assay (*P < 0.01, vs. blank group; and (c) the corresponding values of alpha-fetoprotein proteins at 48.72 h after transfection (*P < 0.01, vs. blank group). Data from HepG2 cells were expressed as a mean ± standard deviation; (d) data from Huh7 cells were expressed as a mean ± standard deviation (*P < 0.01, vs. blank group)

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Compared to the blank control, the results obtained from ELISA assay (n = 3) of supernatant also demonstrated that AFP protein secretion was also significantly reduced after transfection of the stealth RNAi (HSS303, HSS304, and HSS305) and the RNAi cocktail for 48 h and 72 h; the cocktail had the most potent effect. In contrast, transfection of negative control had no obvious silencing effect on secretion of AFP protein. The corresponding values of AFP protein reduction for blank control, negative control, HSS303, HSS304, HSS305, and RNAi cocktail groups at 48 and 72 h after stealth RNAi transfection were shown in [Figure 2]c. This was as same as the results came from Huh7 cells in our previous study[22] [Figure 2]d. Distinct differences were seen between the blank control and RNAi groups. The cocktail of the three stealth RNAi showed the most effective inhibition at 72 h after stealth RNAi transfection ([Figure 2]b, P < 0.01).

Knockdown of alpha-fetoprotein gene decreases HepG2 cell viability

To determine whether knockdown of AFP gene affects cell proliferation in HepG2 cells, metabolic activity at 24, 48, and 72 h after stealth RNAi transfection was determined by MTT assay. The cell viability was reduced significantly after treatment with the stealth RNAi against AFP (HSS304) and the RNAi cocktail at 24, 48, and 72 h as compared with the negative control or blank control ([Figure 3]a, P < 0.05). The cocktail of the three Stealth RNAi had the most potent suppression (P < 0.01). The inhibition rates of cell proliferation were 16.62%, 21.56%, and 21.44% after 24, 48, and 72 h of transfection, respectively. In contrast, transfection of the negative control had no obvious effect on cell proliferation. The corresponding values for blank control, negative control, HSS304, and the RNAi cocktail groups after 24, 48, and 72 h of transfection were shown in [Figure 3]b.
Figure 3: Silencing alpha-fetoprotein gene affects cell proliferation and cell cycle in HepG2 cells. (a) The growth curve was determined by the dimethylthiazolyl-2,5-diphenyl-tetrazolium bromide assay at 24, 48, and 72 h after transfection. (b) The values of their relative absorbance were calculated in terms of the percentage of the blank control. The relative OD values of 490 nm are presented as a mean ± standard deviation (n = 6) (*P < 0.05 compare with blank group). (c) The stealth RNAi against alpha-fetoprotein (HSS304) and cocktails caused accumulation in the G0/G1 phase and decreased the number of cells of S phase at 48 h after stealth RNAi transfection (Figure 3c and d, *P < 0.01)

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Knockdown of alpha-fetoprotein gene affects cell cycle distribution

AFP protein has been reported to enhance the aberrant progression of cancer cells in lower concentration (<0.1 mg/L).[11],[19] In our study, in comparison with nonsilencing siRNA group and negative control, the stealth RNAi against AFP (HSS304) and the RNAi cocktail caused cell accumulation in the G0/G1 phase, and decreased cell numbers in S phase and G2 phase (P < 0.01) at 48 h after stealth RNAi transfection [Figure 3]c and [Figure 3]d.

Knockdown of alpha-fetoprotein gene induces apoptosis in HepG2 cells

To assess whether knockdown of AFP gene could also affect cell survival, we performed Hoechst 33258 staining. Results showed that the cells transfected with HSS304 and the RNAi cocktail displayed obviously chromatin condensation, and nuclear staining was much brighter than it was in the blank control cells [Figure 4]a. In order to further investigate the apoptotic inducing effect of silencing AFP expression in HepG2 cells, we classified and counted apoptotic cells at the early stage, terminal stage, and necrotic stage, respectively, by flow cytometry using double staining with FITC/PI [Figure 4]b. After stealth RNAi transfection for 48 h and 72 h, the apoptotic cells were markedly increased in HSS304 and the cocktail groups compared to those in the negative and blank control groups. Their values were shown in [Figure 4]c (P < 0.01). The results showed that early stage apoptotic cells were markedly increased compared to terminal-stage cells after 48 h transfection, and the apoptotic cells were mainly at the early apoptotic stage after 72 h transfection, which was more significant in the cocktail treated cells.
Figure 4: Silencing alpha-fetoprotein gene can induces apoptosis in HepG2 cell. (a) The cells undergoing apoptosis were visualized with Hoechst 33258 taining; (b) representative analysis by two-parameter Annexin V-fluorescein isothiocyanate/propidium iodide flow cytometry of HepG2 cells at 72 h after stealth RNAi transfection. The proportion of fluorescein isothiocyanate+/propidium iodide− cells corresponds to early apoptosis and that of fluorescein isothiocyanate+/propidium iodide+ cells corresponds to late apoptosis or secondary necrosis; (c) apoptotic ratio were presented as a mean ± standard deviation (*P < 0.01 vs. blank group)

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Effect of knockdown of alpha-fetoprotein gene on transforming growth factor-β and apoptosis-related molecules in HepG2 cells

Although AFP mainly distributed in the supernatant as a secreting protein, we still detected its expression in the cytoplasm by Western blot when analyzing the effect of silencing AFP on the expression of other genes we were interested in. The results showed that AFP protein was nearly absent in HSS304 and the cocktail groups compared with it in negative control groups at 48 h and 72 h after stealth RNAi transfection [Figure 5]a. The previous investigation has demonstrated that interaction between p53 and TGF-β pathways which target chromatin modification and transcription repression of the AFP gene.[31] In order to explore the possible mechanism underlying the cell cycle distribution and apoptosis induction after silencing of AFP in HepG2 cells, further verify whether silencing of AFP expression with AFP stealth RNAi has some effects on TGF-β and P53, we performed Western blot analysis for TGF-β, P53, Bax/Bcl-2, and procaspase-3. Results revealed that decreased expression of AFP can inhibit the expression of TGF-β and induce the expression of caspase-3, the final executor of cell apoptosis. It has been assumed that accumulation of P53 protein represents P53 dysfunction because missense mutations of P53 stabilize the protein by increasing its half-life considerably compared with the wild-type protein,[32] thus the accumulated P53 protein that was detected in this study was therefore assumed to be stabilized by mutation. We found that mutant P53 protein decreased significantly in HSS304 and the cocktail groups compared to the negative control groups at 48 h and 72 h after stealth RNAi transfection (P < 0.05); however, Bax/Bcl-2 ratio was increased in HSS304 and cocktail groups, it was 4.37, 8.66 fold (at 48 h) and 5.09, 8.14 fold (at 72 h) higher, respectively, than that of the negative control group after stealth RNAi transfection ([Figure 5]b, P < 0.01). Therefore, silencing of AFP gene expression inhibited cell proliferation and induced apoptosis in HepG2 cells. The effects may be mediated by the dysfunction of TGF-β or P53/Bax/caspase-3 signaling pathway.
Figure 5: Western blot detects the expression of alpha-fetoprotein, transforming growth factor-β, P53, Bcl-2, Bax, and procaspase-3 in HepG2 cells at 48, 72 h after transfection. (a) The bands shown here are from a representative experiment repeated three times with similar results; (b) the bands were quantified by densitometry using software Glyko Bandscan 5.0 (Glyko Inc., USA), and proteins level were normalized against those of β-actin respectively, data were expressed as a mean ± standard deviation one-way ANOVA followed by Bonferroni correction was used to compare the data among three groups (*P < 0.05 vs. blank group)

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 > Discussion Top


Accumulated data from an investigation of the biological function of AFP indicate it has a close association with the occurrence of HCC. However, the intracellular mechanism of AFP action is still unclear. AFP has pleiotropic properties, which include activities affecting cell growth or apoptosis and tumorigenesis.[18] However, whether AFP may be considered to be a product of a novel tumor gene remains controversial. Recent studies demonstrated a possible correlation between the transcription expression of the AFP gene and the growth of tumor cells. In these studies, p53 protein acting as a transcription repressor was able to bind to the regulatory element of AFP gene, thereby resulted in chromatin modification and inhibition of AFP gene expression, thus implied the relation of over-expression of AFP to over-growth of liver tumor.[31],[33] In order to confirm the pathological role of AFP in tumorigenesis, recent investigations at the molecular level have focused in detail on the mechanism which accounts for the role of AFP on tumor progression and resistance to chemotherapy.[31],[34] Shedding light on these questions may help to advance biological understanding of the therapeutic potential of gene-targeting in greater depth in liver tumors. The data from most current research demonstrates AFP as an enhancer of tumor growth. The downregulation of AFP is able to suppress the growth of malignant hepatocyte cells.[13],[17],[35] On the other hand, high levels of AFP in the fully developed HCC, or in the serum of the host, are associated with more aggressive behavior and increased anaplasia.[36] Therefore, these studies suggest that AFP functions to constitute one of the fundamental steps in the progression of hepatoma. However, the precise mechanism of AFP-mediated cell growth regulation and apoptosis inhibition remains to be elucidated. Revealing the intracellular mechanisms underlying the growth regulation and apoptosis inhibition will provide further insights into the understanding of the biological role of AFP, particularly in the case of HCC.

In recent years, RNAi has been applied to silence specific genes efficiently. It can be used as a powerful tool for investigating genes' function and genetic therapy for carcinoma.[37],[38] To explore the possible role of AFP in tumorigenesis and tumor progression, we silenced AFP expression in HCC cell line HepG2 by using RNAi in the present study. In order to validate Stealth RNAi efficiency, we measured each oligomer's effects on both AFP mRNA and protein levels. The results demonstrated that the stealth RNAi against AFP (HSS303, HSS304, and HSS305) and the cocktail of the three Stealth RNAi can silence AFP expression effectively at both mRNA and protein levels in HepG2 cell after stealth RNAi transfection. In addition, MTT assay confirmed that the proliferation of HepG2 cells was reduced significantly after AFP gene was silenced by RNAi. Flow cytometry also found that in comparison to nonsilencing siRNA group and negative control, the stealth RNAi against AFP (HSS304) and the cocktail caused cell cycle arrest, cell accumulation in G0/G1 phase, and decreased cell numbers in S phase and G2 phase at 48 h after stealth RNAi transfection. Furthermore, using FACS methods (double staining with FITC/PI), we observed that in comparison with nonsilencing siRNA group and blank control, the stealth RNAi against AFP (HSS304) and the cocktail caused accumulation of early stage apoptotic cells at 48 h and 72 h after stealth RNAi transfection (AFP was silenced). The presence of nuclear chromatin condensation and apoptotic bodies in these cells was also confirmed by Hoechst 33258 staining.

Based on the above results, it is clear that AFP may play an important role in the proliferation and apoptosis resistance of hepatoma cells which produce high levels of AFP. Although high concentrations of AFP or AFP-derived peptides have displayed some growth inhibitory properties,[16] AFP concentration as low as below 100 μg/ml in HepG2 cells and most HCC patients could still exhibit apoptosis inhibition. Wang et al.[12] studied in vitro effects of a hairpin AFP-siRNA expressing plasmid on AFP expression in SMMC-7721 cells, and reported that the AFP-siRNA expressing plasmid down-regulated the expression of AFP about 34%, and inhibited SMMC-7721 cell proliferation without inducing apoptosis. As they did not knockdown AFP expression efficiently, we believe that the remaining low concentration of AFP may still have some apoptosis inhibition effect. In our study, we silenced expression of AFP almost completely (81.5%) with transfection of the cocktail of the three stealth RNAi at 72 h, and we did detect the overt apoptosis in AFP silenced cells, even in HSS303 treated cells with 72.1% reduction of AFP protein. These results are as same as that we obtained before from Huh7 cells, another HCC cell line with high AFP level.[22]

The expression of TGF-β is often upregulated in transformed hepatocytes. In fact, it has been reported that plasma TGF-β level was increased in HCC patients, especially during angiogenesis in HCC and could be considered as a marker for HCC progression.[39],[40] Most hepatocarcinoma cells are able to synthesize and secrete TGF-β continually by themselves. Thus, while TGF-β signaling is tumor-suppressive in various tissues, HCC cells often retain sensitivity to TGF-β and possess a functional autocrine TGF-β loop. Recently, researchers found that many of HCC cell lines, including SNU423, HepG2, Sk-Hep-1, and Huh7 cells have an operational TGF-β/Smad signaling pathway and are growth inhibited by exogenous TGF-β1 to varying degrees in both two dimensional and three dimensional growth conditions. However, after autocrine TGF-β signaling is abrogated by the TGF-β receptor, Type II (Tgfbr2) knockdown in SNU423 and Sk-Hep-1 cells, resulted in a significant inhibition of the growth and stimulation of apoptosis in both of cells.[41] Another study also showed that deletion of the Tgfbr2 in the setting of p53 loss reduced the formation of liver tumors in mice with increased levels of AFP, suggesting that TGF-β signaling was playing a promoting role in HCC with high levels of AFP in the setting of p53 loss.[42] These results indicate that the autocrine TGF-β signaling does not inhibit the proliferation, instead is necessary for the viability of these HCC cells. In addition, the data from pancreatic β-cell showed that TGF-β signaling regulates proliferation through control of cell cycle regulator p27 expression. Inhibition of TGF-β signaling reduces the expression of p27, and as a result this inhibition promotes β-cell proliferation.[43]

The wild-type p53 is a very important tumor-suppressor gene for the inhibition of tumor cell proliferation. Some studies have shown that HCC is associated with abnormal expression of p53 gene.[44],[45] Functional inactivation of p53 is often a critical step in the pathway to tumorigenesis. When p53 gene is mutated, it cannot suppress tumor cell growth; on the contrary, it works as an oncogene. Furthermore, mutant p53 can bind with the wild-type p53 resulting in loss of its activity. The previous investigation demonstrates that interaction between p53 and TGF-β pathways targets chromatin modification and transcription repression of the AFP gene.[31] Aberrant expression of AFP during tumorigenesis may require functional inactivation of p53 protein.[46]

In order to explore the possible mechanism underlying the cell cycle arrest, growth inhibition, and apoptosis induction through silencing AFP in HepG2 cells and to verify whether silencing AFP expression affects TGF-β and mutant P53 expression, Western blotting for TGF-β and mutant P53 were performed. The results revealed that silencing AFP expression reduced the expression of TGF-β and mutant P53 protein in HepG2 cells. The downregulation of TGF-β and mutant P53 should not be the off-target effect of the RNAi because the three stealth RNAi used is not homologous to any known genes, and the negative control siRNA does not affect the expression of TGF-β and mutant P53. It suggests that silencing AFP may inhibit the expression of TGF-β and mutant p53 proteins in HepG2 cells. Previous investigation has demonstrated that interaction between p53 and TGF-β pathways targets chromatin modification and transcription repression of the AFP gene.[31] We conclude that the role of AFP, as a growth stimulator and apoptosis inhibitor of HCC, may depend on the interaction with p53 and TGF-β. But the specific mechanism underlying it remains to be determined.

P53 may induce cell apoptosis by transcriptionally down-regulating Bcl-2 and up-regulating Bax expression,[47],[48] which has been suggested to determine the survival or death of the cells following an apoptotic stimulus.[49] Bcl-2 is an upstream effector molecule in the apoptotic pathway and a potent suppressor of apoptosis. It has been shown to form a heterodimer complex with the proapoptotic member Bax, thereby neutralizing its proapoptotic effects. Low expression of the apoptosis inducer Bax correlates with poor response to chemotherapy and shorter overall survival in solid tumors. Since the ratio of Bax/Bcl-2 is important in determining whether the cells will undergo apoptosis or survival,[50] we then determined whether Bax and Bcl-2 are regulated by mutant p53 proteins in HepG2 cells after AFP is silenced by the stealth RNAi. By immunoblotting, we demonstrated that silencing AFP may reduce the expression of mutant p53 (in a direct or indirect way), leading to a decrease in the expression of Bcl-2 but an increase in the expression of Bax. The ratio of Bax/Bcl-2 in HepG2 cells was therefore changed which is similar to that of a tocotrienol-rich fraction of palm oil mediated induction of apoptosis.[51]

During apoptosis, Bcl-2 family proteins regulate the release of mitochondrial apoptotic molecules such as apoptosis inducing factor, and they also activate caspases. We used Western blotting to determine the expression of procaspase-3, the precursor of caspase-3. The results showed that silencing of AFP in HepG2 cells (with HSS304 or the RNAi cocktail) is accompanied by activation of caspase-3, as evidenced by reduced procaspase-3. We propose that AFP may negatively regulate Bax/Bcl-2 ratio by modulating p53 and leads to apoptosis resistance in tumorigenesis and tumor progression of HCC. Hence, by silencing the expression of AFP, Bax/Bcl-2 ratio is upregulated via reducing mutant p53 protein expression which further activates caspase-3 to induce apoptosis. Our findings together with a previous report[52] support a model in which physiological relevant concentrations of AFP play a protective role against apoptosis.

In a word, our study demonstrates that (1) stealth RNAi against AFP can knockdown AFP gene effectively at both mRNA and protein levels in HepG2 cells; (2) knockdown of AFP gene causes cell cycle arrest, inhibits cell growth, and induces apoptosis in HepG2 cells; (3) knockdown of AFP gene inhibits cell growth possibly through the TGF-β signaling pathway in HepG2 cells; and (4) knockdown of AFP gene induces apoptosis possibly through the p53/Bax/caspase-3 apoptosis signaling pathway in HepG2 cells, which needs further studies to confirm. These results imply that AFP may function as a hepatoma growth stimulator or apoptosis inhibitor. With dual roles in promoting cell proliferation and preventing apoptosis, AFP is considered to be a protein that stands at the interface of life and death. It has the potential both as a prognostic marker for HCC and as a target for chemotherapy. Therefore, it is presumed that the suppression of AFP protein expression will improve the therapy efficiency in HCC patients with high AFP expression.

Financial support and sponsorship

The National Natural Science Fund from China (No. 81260326, to Xiaojun Yang).

Conflicts of interest

There are no conflicts of interest.



 
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